Feng Ni

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N′‐acetyl [D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow‐tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α‐thrombin (K i=3.7±0.3 × 10−10 M) and bovine α‐thrombin (1.8±0.7 × 10−9 M). P79 showed saturable inhibition of plasma APTT. The P1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site‐directed tripeptide [D‐Phe‐Pro‐ArgΨ(COCH2)CH3COOCH3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α‐thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α‐thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α‐thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???‐acetyl[D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH2‐ and COOH‐terminal recognition sites.

The NMR solution structure of the NMDA receptor antagonist, conantokin‐T, in the absence of divalent metal ions

The solution conformation of conantokin‐T, a Gla‐containing 21‐residue peptide, (G1EγγY5QKMLγ10NLRγA15EVKKN20A‐amide), in the absence of divalent metal ions, was studied by use of two‐dimensional proton NMR spectroscopy. The peptide is helical from the N‐terminus to the C‐terminus, as defined by upfield‐shifted α‐proton resonances and by characteristic NOE connectivities. Extensive interactions among the amino acid side‐chains were identified from the NOESY spectra of this peptide in a buffered aqueous solution. Four hydrophobic residues Tyr5, Met8, Leu9, and Leu12 contact one another in a stable cluster, even in the presence of 6 M urea. The solution structure of conantokin‐T is a well‐defined α‐helix, having RMSD values for the backbone and all heavy atoms of 0.40 Å and 0.77 Å, respectively. Potential repulsion between the negatively‐charged side chains of Gla10 and Gla14 is minimized by a Gln6‐Gla10 hydrogen bond and by an Arg13‐Gla14 ion‐pair interaction. The C‐terminal amide and the Asn20 side‐chain amide both interact with the backbone and minimize fraying at the C‐terminal end of the α‐helix. This study provides a basis to evaluate the changes in peptide conformation concomitant upon the binding of divalent metal ions. In addition, this investigation demonstrates that apo‐conantokin‐T has almost all of the favorable interactions that are known to contribute to helical stabilization in proteins and monomeric helices.

The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral‐pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys‐29 at the active site and a mutant with Cys‐29 replaced by Ala, were shown to have well‐folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three‐dimensional structure at neutral pH conditions. In addition, replacement of Cys‐29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild‐type cathepsin B. These results are very surprising in that replacement of one single residue, the active‐site Cys‐29, can eliminate the neutral‐pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH‐triggered inactivation commonly observed for most lysosomal cysteine proteases.

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide

Selected fragments of the 62‐residue proregion (or residues 1p–62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p–51p and 26p–51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p–51p fragment, but remained in the 26p–43p and 28p–43p peptide fragments. Residues 23p–26p or TWQ25A in the N‐terminal 1p–29p fragment did not show cathepsin B‐induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p–51p peptide. Both the 1p–29p and 36p–51p fragments lack a common set of hydrophobic residues 30p–35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p–51p, 26p–51p, 28p–43p and 26p–43p, but the same residues induce the aggregation of peptides 16p–36p and 1p–36p. The peptide fragment 26p–43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p–43p or 26p–43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full‐length cathepsin B proregion.